Content uploaded by Moustapha Kassem
Author content
All content in this area was uploaded by Moustapha Kassem
Content may be subject to copyright.
Biogerontology 2: 165–171, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands. 165
Research article
Adipocyte tissue volume in bone marrow is increased with aging and in
patients with osteoporosis
J. Justesen1, K. Stenderup1, E.N. Ebbesen2, L. Mosekilde2, T. Steiniche3& M. Kassem1,∗
1University Department of Endocrinology and Metabolism, Aarhus Amtssygehus, DK-8000, Aarhus-C, Denmark;
2Department of Cell Biology, Instistute of Anatomy, University of Aarhus, DK-8000, Aarhus-C, Denmark;
3University Institute of Pathology, Aarhus Kommunehospital, DK-8000, Aarhus-C, Denmark; ∗Author for
correspondence (e-mail: mkassem@dadlnet.dk)
Received 11 December 2000; accepted in revised form 12 February 2001
Key words: adipose tissue volume, aging, bone, bone biopsies, histomorphometry, human, osteoporosis
Abstract
Aging of the human skeleton is characterized by decreased bone formation and bone mass and these changes
are more pronounced in patients with osteoporosis. As osteoblasts and adipocytes share a common precursor
cell in the bone marrow, we hypothesized that decreased bone formation observed during aging and in patients
with osteoporosis is the result of enhanced adipognesis versus osteoblastogenesis from precursor cells in the
bone marrow. Thus, we examined iliac crest bone biopsies obtained from 53 healthy normal individuals (age
30–100) and 26 patients with osteoporosis (age 52–92). Adipose tissue volume fraction (AV), hematopoietic
tissue volume fraction (HV) and trabecular bone volume fraction (BV) were quantitated as a percentage of
total tissue volume fraction (TV) (calculated as BV + AV + HV) using the point-counting method. We found
an age-related increase in AV/TV (r= 0.53, P< 0.001, n= 53) and an age-related decline in BV/TV (r=
–0.46, P< 0.001, n= 53) as well as in the HV/TV (r= –0.318, P< 0.05, n= 53). There was an age-related
inverse correlation between BV/TV and AV/TV (r= –0.58, P< 0.001). No significant correlation between the
AV/TV and the body mass index (r= 0.06, n.s., n= 52) was detectable. Compared with age-matched controls,
patients with osteoporosis exhibited an increased AV/TV (P< 0.05) and decreased BV/TV (P< 0.05) but no
statistically significant difference in HV/TV. Our data support the hypothesis that with aging and in osteoporosis an
enhanced adipogenesis is observed in the bone marrow and that these changes are inversely correlated to decreased
trabecular bone volume. The cellular and molecular mechanisms mediating these changes remain to be determined.
Introduction
Human aging is associated with a progressive decline
in bone mass and an increased susceptibility for
fractures (Riggs and Melton 1986). The mechan-
isms of age-related bone loss are not known in any
detail. However decreased bone formation, increased
bone resorption, or both have been implicated (Parfitt
1990). Histomorphometric studies performed on iliac
bone biopsies have demonstrated that bone volume
decreases with age both in males and females
(Brockstedt et al. 1993; Lips et al. 1978; Krag-
strup et al. 1983; Melsen et al. 1978; Mosekilde
and Mosekilde 1988; Thomsen et al. 1998) which
may be attributed to an age-related impairment of the
function of the bone forming cell (osteoblasts) (Parfitt
1990).
Osteoblasts and adipocytes share a common
precursor cell present in bone marrow stroma (termed
mesenchymal stem cells [MSCs]) (Pittenger et al.
1999; Rickard et al. 1996). In human MSC cultures,
differentiation into osteoblastic or adipocytic cells is
possible through manipulation of culture conditions
(Park et al. 1999; Rickard et al. 1996). However, the
166
relationship between the osteoblastic and adipocytic
differentiation is not known.
In the present study, we examined the hypothesis
that decreased bone formation observed during aging
is the result of enhanced adipogenesis versus osteo-
blastogenesis leading to decreased bone mass. We
also examined these changes in patients with osteo-
porosis (OP). We quantitated the amount of adipose
tissue, trabecular bone, and hematopoietic tissue in
bone biopsies obtained from a large number of normal
donors and from patients with OP.
Materials and methods
Study subjects
Bone autopsies were obtained from the iliac crest of 53
control individuals of different ages (mean age = 54;
range 30–100 years; 31 females, 22 males). All the
subjects had died in accidents or from acute diseases
without periods of immobility. Subjects with any
history of renal, hepatic or metabolic bone diseases
were excluded from the study as well as subjects with
a history of alcohol or drug abuse. None of the control
subjects had suffered osteoporotic fractures. In addi-
tion, iliac bone biopsies were obtained from 26 female
patients with OP that were diagnosed by the presence
of at least one low energy fracture of the spine (mean
age = 70; range 52–92; 26 females [2 autopsies and 24
biopsies]).
Biopsy preparation
Transcortical iliac crest bone biopsies were obtained
from a standard location 2 cm behind and below the
anterior superior iliac spine. Undecalcified sections
embedded in methylmethacrylate were prepared by
a standard procedure as described previously (Stein-
iche et al. 1992). Seven–eight µm thick sections were
stained in Goldner trichrome for light microscopy.
Counting method
Quantitation of adipocyte tissue volume fraction per
total volume fraction (AV/TV), hematopoietic tissue
volume fraction (HV/TV), and trabecular bone volume
fraction (BV/TV) was performed as described previ-
ously (Kerndrup et al. 1980). In brief, a grid
was projected randomly on the bone sections by a
computer program and the number of points hitting
fat, bone, and hematopoietic marrow were counted
(Figure 1). Any point hitting an artifact or sinusoid
was not considered in the total calculation. The total
volume fraction (TV) refers here to the sum of AV, HV
and BV. Extensive preliminary experiments showed
that for each individual 3 different sections and 360
points per section (36 points in 10 microscopic fields)
needed to be counted in order to obtain reprodu-
cible measurements (Justesen 1999). All sections were
measured without prior knowledge of the age or the
disease state of the subjects. Inter-observer and intra-
observer coefficient of variation for AV/TV measure-
ments were 9% and 6%, respectively.
Statistics
Differences between groups were analyzed using
Mann–Whitney Rank Sum Test and Student’s t-test.
Relationship between variables was determined using
simple regression analysis. Comparisons of slopes
and intercepts were performed by z-test. Results are
expressed as mean ±standard error of the mean
(SEM).
Results
Age-related changes in bone marrow composition
We studied bone autopsies from 53 normal controls
(age 30–100, female = 31, male = 22). AV/TV
increased with age from 40% at the age of 30 to 68%at
age 100 (r= 0.53, P< 0.001) (Figure 2A). In contrast,
BV/TV decreased from 26% at the age of 30 to 12%
at the age of 100 (r= –0.46, P< 0.001) (Figure 2B).
A similar decrease in the HV/TV was observed with
age from 34% at 30 years to 20% at 100 years (r=
–0.32, P< 0.05) (Figure 2C). Furthermore, we found
an age-related inverse correlation between AV/TV and
BV/TV (r= –0.58, P< 0.001) (Figure 3).
Age-related differences in bone marrow composition
in males and females
As the group of normal controls consisted of both
males (n= 22) and females (n= 31), we compared
marrow composition between the two sexes. We found
similar age-related changes in A/TV, BV/TV and
HV/TV in both sexes and there was no difference in
the slope or the intercept of regression lines in any of
these parameters (Figures 4A, B).
167
Figure 1. Example showing projection of grid upon a bone section (×10 objective). A grid was projected randomly on the bone sections by a
computer program and the number of points hitting fat, bone, and hematopoietic marrow were counted.
Correlation between body mass index (BMI) and
A/TV in the bone marrow
In order to determine the physiological regulation of
marrow adipose tissue volume, we examined the rela-
tionship between AV/TV and body weight or BMI
in normal controls. The AV/TV was not significantly
correlated with body weight or BMI (r= 0.06, n.s.)
(data not shown) suggesting that marrow adipocytes
are not directly involved in the overall fat meta-
bolism. This observation was still true when male
(r= 0.15, n.s.) and female (r= 0.10, n.s.) controls
were examined separately and there was no statistic-
ally significant differences in the slope (P= 0.38) or
the intercept (P= 0.32) of the regression lines obtained
in the two sexes (data not shown).
Changes in bone marrow composition in patients with
osteoporosis
We compared marrow composition in a group of
patients with OP and age-matched controls (Table 1).
AV/TV was increased in OP compared with the
controls (63 ±3% for OP versus 55 ±3% for controls,
P< 0.05) and BV/TV was significantly decreased in
OP compared with the controls (14 ±2% for OP
compared with 19 ±1% for controls, P< 0.05). On
the other hand, HV/TV was not changed in OP (22 ±
2% versus 26 ±2% for controls, n.s.). Similar results
were obtained if patients with OP were compared to
age-matched normal female controls (Table 1).
Discussion
In the present study we examined the effect of aging
and osteoporosis on the composition of bone marrow
in iliac crest bone biopsies obtained from a large
sample of males and females of different ages and
from patients with OP. Our results demonstrated
that with aging adipogenesis was enhanced and this
was associated with decreased osteogenesis. These
changes were more pronounced in patients with OP.
We found that with aging AV/TV increased and
this increase was similar in men and women. Our
168
Figure 2. Age-related changes in bone marrow composition
measured in iliac bone biopsies. AV – adipose tissue volume,
TV – total tissue volume, BV – trabecular bone volume, HV –
hematopoietic tissue volume.
Figure 3. Age-related correlation between trabecular bone volume
(BV/TV) and adipose tissue volume (AV/TV) in 53 donors aged
30–100.
results corroborate previous findings reporting a
similar positive correlation between aging and bone
marrow adipose tissue (Burkhardt et al. 1987; Meunier
et al. 1971). Age-related increase in adipogenesis was
also reported in rabbits (Bigelow and Tavassoli 1984)
and in a murine model for accelerated senescence:
SAMP6 mice (Kajkenova et al. 1997) suggesting that
it may be a general phenomenon associated with aging
in different species.
AV/TV was increased in patients with OP
compared to age-matched controls. Two previous
studies have reported an increase in AV/TV in patients
with OP. In the study by Meunier et al (Meunier et
al. 1971), the increase in adipose tissue volume was
only apparent in patients with OP below 65 years.
Burkhardt et al (Burkhardt et al. 1987) found an
increase in adipose tissue volume only in younger
patients with OP aged 27–52 years. As we studied
a large number of patients with OP and age-matched
controls, our results demonstrate that increased adipo-
genesis in bone marrow is associated with the OP
phenotype of whatever age and represents a more
pronounced expression of the physiological age-
related changes. The fact that Meunier et al. (Meunier
et al. 1971) did not detect increased AV/TV in older
patients with OP might be due to the small sample size
of controls.
We found a negative correlation between AV/TV
and BV/TV during aging suggesting that an inverse
relationship exists between these two differentiation
pathways. This is supported by the findings that accel-
169
Table 1. Comparison of tissue composition between patients with
osteoporosis and age-matched controls.
Control OP P1P2
Females Males Combined Females
Number 24 8 32 26
Age 67 ±358±465±370±3 n.s. n.s.
BMI 26 ±226±126±123±4 n.s. n.s.
AT/T V( %) 5 4 ±356±455±363±3 0.02 0.02
BV/TV(%) 19 ±216±219±114±2 0.02 0.03
HV/TV(%) 26 ±328±427±222±2 n.s. n.s.
BMI – body mass index, OP – Patients with osteoporosis, P1–for
comparison between age-matched controls of both sexes and OP
females, P2– for comparison between OP females and age-matched
control females.
erated bone loss observed under certain clinical condi-
tions, e.g., during glucocorticoid treatment (Kawai
et al. 1985; Wang et al. 1977) and postovariec-
tomy (Martin et al. 1990; Martin and Zissimos 1991)
was also associated with increased adipogenesis and
decreased osteogenesis in the bone marrow. In our
studies we measured changes in volume of adipose
tissue which is dependent on adipocyte cell size and
cell number. In a previous stereological study, Rozman
et al. (1989) found that the age-related increase in
marrow adipose tissue volume was due to increased
in adipocyte cell size as well as cell number. While
some in vitro studies employing MSC cultures have
demonstrated that enhanced adipocyte differentiation
was associated with decreased osteoblast differenti-
ation (Bennett et al. 1991; Beresford et al. 1992) the
effect of aging and osteoporosis on the differentiation
potential of MSC in vitro have not been studied in
humans.
The cellular and molecular mechanisms under-
lying age-related changes in adipocyte cell volume
and trabecular bone volume are not known. Burkhardt
et al. (1987) suggested that age-related decrease in
bone marrow vascularity may lead to impaired osteo-
genesis and enhanced adipogenesis due to the effect
of hypoxia. Also, some authors suggested that the
observed age-related increase in adipogenesis could
represent a passive process that fills the space created
by decreased bone mass (Gimble et al. 1996). In
favor of this view are the results of several experi-
mental studies demonstrating that marrow adipocytes
do not play a role in the overall fat metabolism.
For example marrow adipocytes are not affected by
long periods of starvation (Bathija et al. 1979) or
by insulin which is quite different from the changes
Figure 4. Age-related changes in: (A) adipose tissue volume/total
tissue volume (AV/TV) and (B) trabecular bone volume (BV/TV)
with age in males (n= 22) and females (n= 31). Two regression
lines were plotted: grey line for female donors and black line for
male donors. α= slope, β= intercept.
observed in extramedullary adipocytes (Greenberger
1979; Lanotte et al. 1982). Also, in our study we
did not detect significant correlations between adipose
tissue volume in bone marrow and body weight or
BMI.
However, recent studies on MSC biology (Bianco
and Gehron 2000; Nuttall and Gimble 2000; Pittenger
et al. 1999) suggest that the observed age-related
changes in adipogensis and osteoblastogensis are
due to changes in the differentiation potential of
MSC. Differentiation of MSC to a specific lineage in
the bone marrow comprises two processes: cellular
170
commitment to a specific lineage and the prolif-
eration of these lineage-committed cells. Recently,
studies in vitro and in vivo have identified factors that
control lineage-specific commitment. For example
core-binding factor 1 (Cbfa-1) is an essential transcrip-
tion factor for osteoblast differentiation (Ducy et al.
1997). Wnt gene (Ross et al. 2000) and proxisome
proliferator activated receptor γ(PPAR-γ) (Tontonoz
et al. 1994) are important genes for initiation of adipo-
cyte differentiation pathway. Several hormones and
growth factors have been identified as important regu-
lators of adipocyte (Richardson et al. 1992; Carrel and
Allen 2000) and osteoblast cell proliferation (Kassem
1997; Kassem et al. 2000; Langdahl et al. 1998). Some
of these factors exhibit age-related changes (Kveiborg
et al. 2000; Christiansen et al. 2000). However, the
sequential expression of these factors and their relative
contribution to changes in adipogenesisand osteoblas-
togenesis with age and in osteoporosis remain to be
determined.
Acknowledgements
This work was supported by grants from the Danish
Center for Molecular Gerontology, Danish Medical
Research Council, the Novo Nordisk Foundation,
Aage & Johanne Louis-Hansens Memorial Found-
ation, Director E. Danielsen & Hustrus foundation
and the Nordisk Insulin Foundation. The authors
would like to thank Drs. Cecilia Rosada and professor
Leif Mosekilde for comments on the manuscript and
helpful discussions. Professor Flemming Melsen and
Drs Erik F. Eriksen, and Karoline Meldgaard have
kindly provided some of the bone biopsies used in
this study. Ms. Jette Barlach and Anette Baatrup are
thanked for technical assistance.
References
Bathija A, Davis S and Trubowitz S (1979) Bone marrow adipose
tissue: response to acute starvation. Am J Hem 6: 191–198
Bennett JH, Joyner CJ, Triffitt JT and Owen ME (1991) Adipocytic
cells cultured from marrow have osteogenic potential. J Cell Sci
99: 131–139
Beresford JN, Bennett JH, Devlin C, Leboy PS and Owen ME
(1992) Evidence for an inverse relationship between the differen-
tiation of adipocytic and osteogenic cells in rat marrow stromal
cell cultures. J Cell Sci 102: 341–351
Bianco P and Gehron RP (2000) Marrow stromal stem cells. J Clin
Invest 105: 1663–1668
Bigelow CL and Tavassoli M (1984) Fatty involution of bone
marrow in rabbits. Acta Anat (Basel) 118: 60–64
Brockstedt H, Kassem M, Eriksen EF, Mosekilde L and Melsen F
(1993) Age- and sex-related changes in iliac cortical bone mass
and remodeling. Bone 14: 681–691
Burkhardt R, Kettner G, Bohm W, Schmidmeier M, Schlag R,
Frisch B, Mallmann B, Eisenmenger W and Gilg T (1987)
Changes in trabecular bone, hematopoiesis and bone marrow
vessels in aplastic anemia, primary osteoporosis, and old age:
a comparative histomorphometric study. Bone 8: 157–164
Carrel AL and Allen DB (2000) Effects of growth hormone on
adipose tissue. J Pediatr Endocrinol Metab 13(2): 1003–1009
Christiansen M, Kveiborg M, Kassem M, Clark BF and Rattan SI
(2000) CBFA1 and topoisomerase I mRNA levels decline during
cellular aging of human trabecular osteoblasts. J Gerontol A Biol
Sci Med Sci 55(4): B194–200
Ducy P, Zhang R, Geoffroy V, Ridall AL and Karsenty G (1997)
Osf2/Cbfa1: a transcriptional activator of osteoblast differenti-
ation. Cell 89: 747–154
Gimble JM, Robinson CE, Wu X and Kelly KA (1996) The function
of adipocytes in the bone marrow stroma: an update. Bone 19:
421–428
Greenberger JS (1979) Corticosteroid-dependent differentiation of
human marrow preadipocytes in vitro. In Vitro 15: 823–828
Justesen J (1999) Effect of ageing and osteoporosis on adipocyte
cell differentiation in human bone marrow. MS Thesis. Insti-
tute of Molecular and Structural Biology, University of Aarhus,
Denmark
Kajkenova O, Lecka-Czernik B, Gubrij I, Hauser SP, Takahashi K,
Parfitt AM, Jilka RL, Manolagas SC and Lipschitz DA (1997)
Increased adipogenesis and myelopoiesis in the bone marrow of
SAMP6, a murine model of defective osteoblastogenesis and low
turnover osteopenia. J Bone Miner Res 12: 1772–1779
Kassem M (1997) Cellular and Molecular effects of growth
hormone and estrogen on human bone cells. APMIS 105: 1–30
Kassem M, Kveiborg M and Eriksen EF (2000) Production and
action of transforming growth factor-beta in human osteoblast
cultures: dependence on cell differentiation and modulation by
calcitriol. Eur J Clin Invest 30: 429–437
Kawai K, Tamaki A and Hirohata K (1985) Steroid-induced accu-
mulation of lipid in the osteocytes of the rabbit femoral head. A
histochemical and electron microscopic study. J Bone Joint Surg
67A: 755–763
Kerndrup G, Pallesen G, Melsen F and Mosekilde L (1980) Histo-
morphometrical determination of bone marrow cellularity in iliac
crest biopsies. Scand J Haematol 24: 110–114
Kragstrup J, Melsen F and Mosekilde L (1983) Thickness of bone
formed at remodeling sites in normal human iliac trabecular
bone: variations with age and sex. Metab Bone Dis Relat Res
5: 17–21
Kveiborg M, Flyvbjerg A, Rattan SI and Kassem M (2000) Changes
in the insulin-like growth factor-system may contribute to in
vitro age-related impaired osteoblast functions. Exp Gerontol 35:
1061–1074
Langdahl BL, Kassem M, Moller MK and Eriksen EF (1998) The
effects of IGF-I and IGF-II on proliferation and differentiation of
human osteoblasts and interactions with growth hormone. Eur J
Clin Invest 28: 176–183
Lanotte M, Scott D, Dexter TM and Allen TD (1982) Clonal
preadipocyte cell lines with different phenotypes derived from
murine marrow stroma: factors influencing growth and adipo-
genesis in vitro. J Cell Physiol 111: 177–186
Lips P, Courpron P and Meunier PJ (1978) Mean wall thickness of
trabecular bone packets in the human iliac crest: changes with
age. Calcif Tissue Res 26: 13–17
171
Martin RB and Zissimos SL (1991) Relationships between marrow
fat and bone turnover in ovariectomized and intact rats. Bone 12:
123–131
Martin RB, Chow BD and Lucas PA (1990) Bone marrow fat
content in relation to bone remodeling and serum chemistry in
intact and ovariectomized dogs. Calcif Tissue Int 46: 189–194
Melsen F, Melsen B, Mosekilde L and Bergmann S (1978) Histo-
morphometric analysis of normal bone from the iliac crest. Acta
Pathol Microbiol Scand [A] 86: 70–81
Meunier P, Aaron J, Edouard C and Vignon G (1971) Osteoporosis
and the replacement of cell populations of the marrow by adipose
tissue. A quantitative study of 84 iliac bone biopsies. Clin Orthop
Rel Res 147–154
Mosekilde L and Mosekilde L (1988) Iliac crest trabecular bone
volume as predictor for vertebral compressive strength, ash
density and trabecular bone volume in normal individuals. Bone
9: 195–199
Nuttall ME and Gimble JM (2000) Is there a therapeutic oppor-
tunity to either prevent or treat osteopenic disorders by inhibiting
marrow adipogenesis? Bone 27: 177–184
Parfitt A (1990) Bone forming cells in clinical conditions. In: Hall
B (ed) The Osteoblast and Osteocyte, pp 351–429. Telford Press,
UK
Park SR, Oreffo RO and Triffitt JT (1999) Interconversion poten-
tial of cloned human marrow adipocytes in vitro. Bone 24: 549–
554
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R,
Mosca JD, Moorman MA, Simonetti DW, Craig S and Marshak
DR (1999) Multilineage potential of adult human mesenchymal
stem cells. Science 284: 143–147
Richardson RL, Hausman GJ and Gaskins HR (1992) Effect of
transforming growth factor-beta on insulin-like growth factor 1-
and dexamethasone-induced proliferation and differentiation in
primary cultures of pig preadipocytes. Acta Anat (Basel) 145:
321–326
Rickard DJ, Kassem M, Hefferan TE, Sarkar G, Spelsberg TC and
Riggs BL (1996) Isolation and characterization of osteoblast
precursor cells from human bone marrow. J Bone Miner Res 11:
312–324
Riggs BL and Melton LJ (1986) Involutional osteoporosis. N Engl J
Med 314: 1676–1686
Ross SE, HematiN, Longo KA, Bennett CN, Lucas PC, Erickson
RL and MacDougald OA (2000) Inhibition of adipogenesis by
Wnt signaling. Science 289: 950–953
Rozman C, Feliu E, Berga L, Reverter JC, Climent C and Ferran
MJ (1989) Age-related variations of fat tissue fraction in normal
human bone marrow depend both on size and number of adipo-
cytes: a stereological study. Exp Hematol 17: 34–37
Steiniche T, Eriksen EF, Kudsk H, Mosekilde L and Melsen F
(1992) Reconstruction of the formative site in trabecular bone
by a new, quick, and easy method. Bone 13: 147–152
Thomsen JS, Ebbesen EN and Mosekilde L (1998) Relationships
between static histomorphometry and bone strength measure-
ments in human iliac crest bone biopsies. Bone 22: 153–163
Tontonoz P, Hu E, Graves RA, Budavari AI and Spiegelman BM
(1994) mPPAR gamma 2: tissue-specific regulator of an adipo-
cyte enhancer. Genes Dev 8: 1224–1234
Wang GJ, Sweet DE, Reger SI and Thompson RC (1977) Fat-cell
changes as a mechanism of avascular necrosis of the femoral
head in cortisone-treated rabbits. J Bone Joint Surg [Am] 59:
729–735
